Relationship between the Regulator of Calcineurin 1-4 Isoform and In Vitro Osteoclast DifferentiationPark, Kyeong-Lok;

Abstract

Regulator of calcineurin 1 (RCAN1) is an endogenous calcineurin inhibitor that plays an important role in the pathogenesis of diseases related to the calcineurin-NFATc1 signaling pathway. The RCAN1-4 isoform is subject to NFATc1-dependent regulation. During receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated osteoclastogenesis, the calcineurin-NFATc1 pathway is critical. Because there is little information available on the role of RCAN1 in osteoclast differentiation, this study investigated whether changes in RCAN1 expression are related to the calcineurin-NFATc1 pathway and osteoclast differentiation. Mouse bone marrow monocytes (BMMs) were treated with 50 ng/ml of RANKL and M-CSF. Expression levels of NFATc1, calcineurin, and RCAN1 isoforms were determined using RT-PCR and Western blotting. Osteoclast differentiation was examined using tartrate-resistent acid phosphatase (TRAP) staining. To evaluate the effect of RCAN1 overexpression on osteoclastogenesis, cells were transfected with a mouse RCAN1-4 cDNA plasmid. After RANKL stimulation of BMMs, expression of NFATc1 and RCAN1 was increased at the mRNA and protein level, while calcineurin expression was unchanged. When the RCAN1-4 gene construct was transfected, the expression of RCAN1 protein was not increased despite several-fold increases in RCAN1-4 mRNA expression. Regardless of RANKL stimulation, over-expression of RCAN1-4 tended to reduce NFATc1 expression and knock-down of RCAN1 increase it. While BMMs transfected with the RCAN1-4 vector were differentiated into distinct osteoclasts, their phenotypes did not vary from those of mock controls. These results suggest that RCAN1 has a limited effect on the calcineurin-NFATc1 pathway during RANKL-stimulated osteoclast differentiation.